Int. J. DH. BioI. ~2: 783-789 (1998) Oliginal Article Trefoil peptides are early markers of gastrointestinal maturation in the rat MARY FAMILARI*, GREGORY A. COOK, DOUGLAS R. TAUPIN, GINA MARRYATT, NEVILLE D. YEOMANS and ANDREW S. GIRAUD The University of Melbourne, Department of Medicine, Western Hospital, Footscray, Australia ABSTRACT Trefoil peptides are members of a unique family of proteins found predominately throughout the gastrointestinal tract, whose proposed functions include mucus stabilization. stimulation and/or differentiation of epithelial cells during wound repair. Recent trefoil knockout studies have reported delays in epithelial cell migration or maturation pathways together with almost a complete lack of mucus. In order to fully explore the role of trefoil peptides in gastrointestinal maturation, these studies were undertaken to accurately characterize the expression of trefoil peptides in the developing rat gut. The results of RPA suggest that trefoil mRNA's are expressed as early as 15 days post coitus (dpc) in the intestine and stomach. Proteins are detected at 17 dpc by radioimmunoassay and immunohistochemical studies, which localize trefoil peptide expression to the lumenal surface of epithelial cells. At 17 dpc the gut is lined by pseudo-stratified, undifferenti- ated epithelial cells. Polarized, columnar cells are not detected until at least 18 dpc, with sparse mucus staining and parietal cell markers not being detected until 18 and 19 dpc respectively. This data demonstrates that trefoil peptides are early markers of epithelial cell maturation in the developing rat gut. The time course of their expression, well before the mucus cell type is specified, suggests a potential role in epithelial cell differentiation. KEY WORDS: trefoit pejJtides, gut, development Introduction Trefoil peptides are members of a unique family of proteins characterized by containing one or more three. looped structural motifs held together by disulphide bonds between highly con- served cysteine residues. The known members of this family are pS2 (also known as TFF-1), spasmolytic polypeptide (SP, also known as TFF-2) and intestinal trefoil factor (ITF, also known as TFF-3) (Wright et al., 1997). Trefoils are found throughout the adult gastrointestinal (G.I) tract, with pS2 and SP predominately found in the stomach and ITF in the intestine and colon. They are secreted by mucus producing cells of the normal G.I tract and are highly expressed in cells surrounding areas of damage in condi. tions such as peptic ulceration and inflammatory bowel disease (Rio et al., 1991; Wright et al., 1993: Thim, 1994). The function of trefoil peptides is not fully understood, but it has been proposed that in the normal gut the trefoil peptides may playa role in mucus stabilization. In addition, their up regulation at the site of mucosal injury is consistent with a roJe for these peptides in migration or differentiation durin9 the epithelial repair process (Sands and Podolosky 1996: Cook et a/., 1997). The results of gene-targeting experiments in which pS2(Lefebvre et al., 1996) or ITF (Mashimo et al., 1996) genes were made dysfunctional have highlighted the effects of trefoil peptides on migration of epithelial cells. In these studies, the proliferative (stem cell) compartment of the epithelium was greatly expanded in the colon for ITFand antral mucosa for pS2, strongly implying a defect in cell migration or maturation pathways. The latter was especially evident in pS2 knockout mice in which the distal stomach was hyperplastic with inappropriate cellular differentiation patterns and almost entirely devoid of mucus. Many studies have demonstrated the motogenic capacity of recombinant trefoil peptides for different epithelial cell lines (Dignass et al., 1994: Playford et af., 1995) and it is possible that this may be an important function for trefoil peptides in fetal gut development. Very little is known about the molecular mechanisms involved in vertebrate gut development. Reciprocal mesenchymal-epithelial inductive interactions are known to be of great importance in the functional differentiation of distinct regions of the gut (Simon- Assrnann and Kedinger, 1993; Yasugi, 1993). Results of recent studies suggest that Sonic hedgehog-Hox/Bmp genes may be involved in epithelial-mesenchymal interactions in the earliest .Address for reprints: The University of Melbourne, Department of Medicine, Western Hospital, Footscray, 3011, Australia. FAX: 61.3-9318 1157. e-mail: m.familari@medicine.unimelb.edu.au 02 t 4-6282/97/$ 10.00 () UBC Pre" Printed in Spain -----